It is now established that at least three unique cell death pathways contribute to neuronal death after traumatic brain injury (TBI) in experimental models and in humans. These include the necrotic, caspase-dependent apoptotic, and caspase-independent apoptosis-inducing factor (AIF)-mediated pathways. The latter two pathways were the focus of this project during the previous two funding periods. Compelling preliminary data suggest that autophagy may represent an additional, novel cell death pathway after brain injury, and that similar to apoptosis, it is regulated at least in part by oxidative stress after TBI. Autophagy is a homeostatic process by which cells recycle amino acids and other nutrients from proteins and organelles. Also similar to apoptosis, either sub- or supra-physiologic autophagy can result in disease, but its role after acute, non- starvation related brain injury remains undefined. Furthermore, building upon previous studies it was discovered that autophagic stress may be sex-specific both in vitro and in vivo. The hypothesis of the current application is that autophagic stress contributes to neuronal cell death after traumatic injury in vitro, in experimental models, and in humans, and that inhibiting autophagy reduces neuropathologic damage and improves functional outcome after TBI in a sex-specific manner. Specific Aims are: 1) Establish whether autophagic stress-mediated neuronal death can be induced by traumatic injury, if it is dependent or independent of nutrient signals, and whether it is sex-specific;2) Fully characterize autophagy after TBI in vivo;3) Use known inhibitors of autophagy to establish the role of autophagic stress-mediated cell death after TBI in vivo;and 4) Determine whether autophagic stress-mediated cell death occurs in humans after TBI. TBI is a major cause of morbidity and mortality in adults, and is the leading cause of death in children. Delayed cell death contributes to morbidity and mortality and currently no specific therapies have successfully transitioned from experimental studies to the bedside. Work from the previous two funding periods established significant, but only partial roles for caspase-dependent and AIF-mediated cell death after TBI and sex- proclivity in cell death pathways in vitro. Preliminary studies suggest that other unique, non-necrotic pathways may participate and culminate in overall neuronal death after TBI. The proposed experiments address the key question: Does autophagy contribute to delayed neuronal death and cognitive deficits after TBI, or alternatively, does autophagy have a homeostatic role, recycling proteins and injured organelles similar to conditions of starvation? If inhibition of autophagy improves neurological outcome in a sex-specific manner after TBI, and autophagy is confirmed after TBI in humans, novel, sex-based strategies targeting multiple cell death pathways will be developed to be tested alone and in combination after brain injury. PUBLIC HEALTH RELEVANCE: Traumatic brain injury is a major cause of morbidity and mortality in adults, and is the leading cause of death in children. Several unique pathways contribute to overall brain damage. This application seeks to define clinically important pathways, and design novel, sex-based strategies that improve neurological outcome after brain injury.